Bloodstream infection detection directly on whole blood Summary Sepsis is a life-threatening condition triggered by the presence of fungi or bacteria in the bloodstream that affects 1.3 million people each year in the United States.1 Rapid antimicrobial administration is critical for sepsis treatment.2-4 However, the current gold standard for bloodstream infection diagnosis is blood culture (BC), which takes one to five days.5 In order to provide physicians with the needed diagnostic information to properly treat patients, we propose the development of an automated platform for rapid, broad and cost-effective identification of microorganisms in whole-blood without BC. In this proposal, we will focus on the development of the instrument and the first assay which will detect fungi directly on whole-blood. The new platform will be based on Single Molecule Scanning (SMS), a novel sample preparation and molecular detection method developed at Scanogen. The fungi assay (SMS-Fungi) will detect and identify the five most common Candida species that cause bloodstream infections as well as the Cryptococcus genus. The assay will also utilize probes that target broadly conserved sequences to detect the presence of any fungi in the whole- blood specimen. The goal is to develop a 1-hour, accurate, broad, multiplexed, fully-automated and cost-effective assay, that will aid physicians to start proper antifungal therapy without delay. In phase I, we developed an assay protocol capable of detecting fungi directly in whole-blood. We tested both specific and broad-range probes with four species of fungi commonly found in bloodstream infections. We found that the assay is highly specific and has the high sensitivity required for the diagnosis of clinical cases with a limit of detection (LOD) as low as 1 CFU/ml. In this project, we will complete the development of the assay protocol by including new probes and optimizing the overall assay (Aim 1), develop a beta-prototype system consisting of an automated instrument and disposable cartridge (Aim 2) and evaluate the new system in pilot analytical and clinical validation studies (Aim 3). We will leverage the experience attained in the development of a rapid assay for point of care detection of Tuberculosis currently under development at Scanogen, and work with a multidisciplinary team that includes experts in assay development and instrument development including the former Vice President of Engineering at Becton Dickinson, experts in fungal infection diagnosis and treatment from Johns Hopkins University and engineers from Key Technologies. Our goal is to develop and validate an automated molecular platform for rapid analysis of microorganisms in whole-blood and develop its first assay for fungi detection. If successful, the new assay will dramatically improve the management of patients with fungemia by reducing diagnostic delay and enabling timely initiation of proper antifungal treatment.